Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Nfia

Cell type

Cell type Class
Neural
Cell type
Retina
MeSH Description
The ten-layered nervous tissue membrane of the eye. It is continuous with the OPTIC NERVE and receives images of external objects and transmits visual impulses to the brain. Its outer surface is in contact with the CHOROID and the inner surface with the VITREOUS BODY. The outer-most layer is pigmented, whereas the inner nine layers are transparent.

Attributes by original data submitter

Sample

source_name
retina
age
P2
tissue
retina
genotype
Nfia/b/x cKO
chip antibody
NFI (NFIA, NFIB, NFIX)
replicate
1

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin immunoprecipitation was then performed on the sheared DNA using the iDeal ChIP-seq kit for transcription factors (Diagenode Cat# C01010170). One percent of chromatin was kept aside to be used as an input control. Antibodies against targets used for chromatin immunoprecipitation are NFI (NFIA, NFIB, NFIX), and IgG. Briefly, equal volume of sheared chromatin was incubated overnight with 3 μg of antibody in iC1b buffer with protease inhibitors and BSA and washed DiaMag Protein A-coated magnetic beads (Diagenode Cat# C03010020-220) on a tube rotator at 4°C overnight. The magnetic beads were then washed sequentially with wash buffers iW1, iW2, iW3 and iW4. DNA was then de-crosslinked and eluted for 4 hours at 65C before being purified using IPure beads (Diagenode Cat# C03010014). The purified DNA was then subjected to sequencing library preparation or qPCR analysis. Libraries were prepared from 5ng of DNA using the Ovation Ultralow System V2 (Tecan Genomics Cat# 0344NB-32) and sequenced on the Illumina NextSeq500. Dissociated cells were resuspended in ice-cold PBS containing 0.04% bovine serum albumin (BSA). Cell count and viability were assessed by Trypan blue staining.Trypan blue staining. scATAC-seq was performed using the 10x Genomic single cell ATAC reagent v1.1 kit following the manufacturer's instruction. Briefly, dissociated cells were centrifuged at 300xg for 5 min at 4oC. Cell pellet was resuspended in 100 μl of Lysis buffer, mixed 10x by pipetting and incubated on ice for 3 min. Wash buffer (1 ml) was added to the lysed cells, and cell nuclei were centrifuged at 500xg for 5 min at 40C. Nuclei pellet was re-suspended in 250 μl of 1x Nuclei buffer. Cell nuclei were then counted using Trypan blue. Re-suspended cell nuclei (10-15k) were used for transposition and loaded into the 10x Genomics Chromium Single Cell system. Libraries were amplified with 10 PCR cycles and were sequenced on Illumina NextSeq or NovaSeq with ~200 million reads per library. Sequencing data was processed through the Cell Ranger ATAC 1.1.0 pipeline (10x Genomics) using default parameters. single cell ATACseq

Sequencing Platform

instrument_model
NextSeq 500

mm10

Number of total reads
69796058
Reads aligned (%)
97.0
Duplicates removed (%)
16.9
Number of peaks
10199 (qval < 1E-05)

mm9

Number of total reads
69796058
Reads aligned (%)
96.8
Duplicates removed (%)
17.2
Number of peaks
10237 (qval < 1E-05)

Base call quality data from DBCLS SRA